A disposable amperometric biosensor for ketone 3-β-hydroxybutyrate (3HB) has been developed successfully. The sensor is based on a screen-printed carbon electrode containing Meldola’s Blue (MB) and sensing components containing nicotinamide adenine dinucleotide (NAD+) and 3-β-hydroxybutyrate dehydrogenase (3HBDH) immobilized in mesoporous silica (FSM8.0) using an aqueous photo-cross-linkable polymer matrix of polyvinyl alcohol (O-391), and it requires only a small sample volume of 10 μL for the measurement. The behavior of a resulting biosensor, i.e., 3HBDH–FSM8.0/NAD+/MB-SPCE, was examined in terms of NAD+ concentration for construction, pH, applied potential, operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.6 and at an applied potential of −50 mV. The determination range and the response time for 3HB were from 30 μM to 8 mM and approximately 30 s, respectively. In addition, the sensor was quite stable and maintained >90 % of its initial response after being stored for over 6 months. This result implies that our method provides a novel biosensor for ketone 3-β-hydroxybutyrate which is easy-to-use, cost-effective, and has good reproducibility, which are vital for commercial purposes.